Cross-species comparison of CAR-mediated procarcinogenic key events in a 3D liver microtissue model. (2019)

• Record Type: Journal Article
• Authors: Plummer, Simon; Beaumont, Bobby; Wallace, Stephanie; Ball, Graeme; Wright, Jayne; McInnes, Liz; Currie, Richard; Peffer, Rich; Cowie, David
• Is Part Of: Toxicology reports. Volume 6(2019)
• Page Start: 998
• Page End: 1005
• Publication Date: 2019
• Subjects: 3D liver microtissues -- Tissue microarray -- Constitutive androstane receptor -- Key events -- Carcinogenesis -- Quantitative histopathology -- Cross-species risk assessment -- Transcriptomics -- Proteomics -- Hepatocyte -- Mode of action -- Proliferation -- Enzyme induction -- Pathways -- MicroTMA -- Species difference; Toxicology -- Periodicals; Clinical toxicology -- Periodicals; Drug-Related Side Effects and Adverse Reactions; Hazardous Substances; Poisoning; Toxicology; Electronic journals; Periodicals; Periodicals; 571.9505
• DOI: 10.1016/j.toxrep.2019.09.010
• Languages: English
• ISSNs: 2214-7500

Abstract

Highlights: The first demonstration of phenobarbital-induced hepatocyte proliferation in 3D liver microtissue models. Integration of quantitative histopathology data with genomics (transcriptomics/proteomics). In vitro cross-species risk assessment. CAR-mediated mode of action. Quantitative histopathology of 3D microtissues. Abstract: Characterisation of the mode of action (MOA) of constitutive androstane receptor (CAR)-mediated rodent liver tumours involves measurement 5 key events including activation of the CAR receptor, altered gene expression, hepatocellular proliferation, clonal expansion and increased hepatocellular adenomas/carcinomas. To test whether or not liver 3D microtissues (LiMTs) recapitulate CAR- mediated procarcinogenic key events in response to the prototypical CAR activator phenobarbital (PB) we performed hepatocyte proliferation (LI%) analysis in rat and human LiMTs using a microTMA technology in conjunction with integrated transcriptomics (microarray) and proteomics analysis. The rationale for this approach was that LiMTs containing parenchymal and non-parenchymal cells (NPCs) are more physiologically representative of liver and thus would generate data more relevant to the in vivo situation. Rat and human LiMTs were treated with PB over a range of concentrations (500 uM - 2000 uM) and times (24 h - 96 h) in a dose-response/time-course analysis. There was a dose-dependent induction of LI% in rat LiMTs, however there was little or no effect of PB on LI% in human LiMTs. ATP levels in the rat and human LiMTs were similar to control in all of the PB treatments. There was also a dose- and time-dependent PB-mediated RNA induction of CAR regulated genes CYP2B6/Cyp2b2, CYP3A7/Cyp3a9 and UGT1A6/Ugt1a6 in human and rat LiMTs, respectively. These CAR regulated genes were also upregulated at the protein level. Ingenuity pathways analysis (IPA) indicated that there was a significant (Z score >2.0;-log p value >) activation of CAR by PB in both human and rat LiMTs. These results indicate that human and rat LiMTs showed the expected responses at the level of PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing significant dose-dependent effects while human LiMTs showed no proliferation response but did show dose-dependent enzyme induction at the RNA and protein levels. In conclusion LiMTs serve as a model to provide mechanistic data for 3 of the 5 key events considered necessary to establish a CAR-mediated MOA for liver tumourigenesis and thus can potentially reduce the use of animals when compiling mechanistic data packages.